Khoo's Lab - Cellular Glycomics

On the (sulfo)glycomics front, we have focused particularly on sialylated glycans carrying Gal-6-O-sulfate and/or GlcNAc-6-O-sulfate modifications that can markedly enhance recognition by selected Siglecs. This is motivated by the emerging role of Siglecs as inhibitory immune checkpoints in peripheral immunity and in the central nervous system, where altered Siglec–glycan interactions are increasingly implicated in neuroinflammation and neurodegenerative disease (Siew et al., Mol Aspects Med, 2022). In collaboration with the Macauley laboratory, we applied detailed sulfoglycomic profiling to CHST1-overexpressing and glycogene-knockout cell systems to dissect the biosynthetic pathways that generate sulfated Siglec ligands. These studies showed how sulfotransferases, glycosyltransferases and the underlying glycan carriers act combinatorially to generate high-affinity Siglec-binding glycotopes (Jung et al., ACS Chem Biol, 2025).

In parallel, we have advanced negative-ion LC-MS/MS methods for sequencing highly sulfated polyLacNAc and keratan sulfate structures, which are particularly challenging because both chain length and positional sulfation must be resolved. In ovarian carcinoma, our workflow enabled direct characterization of low- and high-sulfated keratan sulfate, including O-glycans containing up to five LacNAc repeats and four sulfate groups, and helped establish structural distinctions associated with tumor subtype and prognosis (Hoshino et al., Glycobiology, 2024; Inoue et al., Lab Invest, 2024, supported by NSTC 112-2311-B-001-014-MY3). The broader utility of this approach was further illustrated in seal milk oligosaccharides, where our permethylation-based MS analysis uncovered exclusively branched polyLacNAc chains capped by either NeuAc or Fuc, extending up to 28 glycosyl residues and revealed that GlcNAc sulfation locally constrains adjacent galactose branching (Jin et al., Nat Comm, 2025). Together, these studies establish sulfoglycomics as a distinctive strength of our laboratory for resolving structural features that are easily missed by conventional glycomic workflows, and position us to connect sulfated glycan architecture with Siglec recognition, carcinoma biology and immune homeostasis across diverse biological systems.

Siew JJ, Chern Y, Khoo KH, and Angata T (2022) Roles of Siglecs in neurodegenerative diseases. Mol Aspects Med, 101141. https://doi.org/10.1016/j.mam.2022.101141
Jung J, Schmidt EN, Chang HC, Jame-Chenarboo Z, Enterina JR, McCord KA, Gray TE, Kageler L, St Laurent CD, Wang C, Flynn RA, Wu P, Khoo KH, and Macauley MS (2025) Understanding the Glycosylation Pathways Involved in the Biosynthesis of the Sulfated Glycan Ligands for Siglecs. ACS Chem Biol 20, 386-400. https://doi.org/10.1021/acschembio.4c00677
Hoshino H, Chen YY, Inoue D, Yoshida Y, Khoo KH, Akama TO, and Kobayashi M (2024) Expression of low-sulfated keratan sulfate in non-mucinous ovarian carcinoma. Glycobiology 34, cwad056. https://doi.org/10.1093/glycob/cwad056
Inoue D, Hoshino H, Chen YY, Yamamoto M, Kogami A, Fukushima M, Khoo KH, Akama TO, Yoshida Y, and Kobayashi M (2024) Structural elucidation and prognostic relevance of 297-11A-sulfated glycans in ovarian carcinoma. Lab Invest, 102057. https://doi.org/10.1016/j.labinv.2024.102057
Jin C, Lundstrom J, Cori CR, Guu SY, Bennett AR, Dannborg M, Pomeroy PP, Kennedy MW, Bengtsson-Palme J, Hevey R, Khoo KH, and Bojar D (2025) Seal milk oligosaccharides rival human milk complexity and exhibit functional dynamics during lactation. Nat Commun 16, 10067. https://doi.org/10.1038/s41467-025-66075-2

Other representative works:

Discovery Sulfoglycomics and Identification of the Characteristic Fragment Ions for High-Sensitivity Precise Mapping of Adult Zebrafish Brain-Specific Glycotopes. (2021). Tseng HC, Hsiao CT, Yamakawa N, Guerardel Y and Khoo KH. Front Mol Biosci 8: 771447.
A mass spectrometry-based glycotope-centric cellular glycomics is the more fruitful way forward to see the forest for the trees. (2021). Khoo KH. Biochem Soc Trans 49(1): 55-69.
Functional roles of ST8SIA3-mediated sialylation of striatal dopamine D2 and adenosine A2A receptors. (2019). Lin CY, Lai HL, Chen HM, Siew JJ, Hsiao CT, Chang HC, Liao KS, Tsai SC, Wu CY, Kitajima K, Sato C, Khoo KH and Chern Y. Transl Psychiatry 9(1): 209.
Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry-based sulfoglycomic analysis. (2018). Yu SY, Hsiao CT, Izawa M, Yusa A, Ishida H, Nakamura S, Yagi H, Kannagi R and Khoo KH. J Biol Chem 293(39): 15163-15177.
Concerted mass spectrometry-based glycomic approach for precision mapping of sulfo sialylated N-glycans on human peripheral blood mononuclear cells and lymphocytes. (2018). Chen JY, Huang HH, Yu SY, Wu SJ, Kannagi R and Khoo KH. Glycobiology 28(1): 9-20.
Advancing a high throughput glycotope-centric glycomics workflow based on nanoLC-MS2-product dependent-MS3 analysis of permethylated glycans. (2017). Hsiao CT, Wang PW, Chang HC, Chen YY, Wang SH, Chern Y and Khoo KH. Mol Cell Proteomics 16(12): 2268-2280.
Efficient mapping of sulfated glycotopes by negative ion mode nanoLC-MS/MS-based sulfoglycomic analysis of permethylated glycans. (2015). Cheng CW, Chou CC, Hsieh HW, Tu Z, Lin CH, Nycholat C, Fukuda M and Khoo KH. Anal Chem 87(12): 6380-6388.